Project Summary RNA modifications are emerging epitranscriptomic markers in regulating gene expression, and are crucial for development and linked to human diseases, such as neuronal disorders and cancers. N6-methyladenosine (m6A) is the most prevalent modification in the messenger RNA (mRNA) and long noncoding RNA (lncRNA) in higher eukaryotes. m6A are dynamically installed and removed on the target RNAs, and can be recognized by various m6A reader proteins (proteins containing YTH domains, or YTH proteins) to regulate the target RNAs through diverse mechanisms, or alter the local RNA structure to fine tune the RNA-protein interactions. How does one single type of modification allow one RNA target to be regulated in diverse ways? What are the signals contributing to the choice of a specific regulatory mechanism for a particular RNA? We hypothesize that a single RNA can exist in multiple ?methylation states? due to the existence of multiple methylation sites and incomplete methylation at each site. The methylation state serves as ?epitranscriptomic code? and may determine fate of the RNA. The methylation states of a single RNA and the mechanistic linkage between methylation states and various m6A-mediated regulation pathways can only be dissected by experimental approaches with both specificity to a defined methylation site and sensitivity of single RNA. However, such experimental approaches are currently unavailable, which hampers our current efforts to understand the role of RNA modifications. To tackle this problem, in Aim 1, we will first develop a suite of imaging platforms with single-RNA sensitivity and single- methylation site specificity. With the unique techniques, we will then focus on studying lncRNA and mRNA systems. In Aim 2, we will investigate whether methylation state of an lncRNA, MALAT1, has impact on its multivalent binding capacity, its subcellular localization, and its recruitment to actively transcribed gene loci to promote splicing. In Aim 3, we will explore how methylation state in an mRNA can influence choice of regulatory pathways at translation or degradation levels, and the strength of m6A-mediate regulation. Our proposed experiments on RNA modification at single-RNA level in vivo will provide valuable mechanistic insight of m6A-mediated regulation in both lncRNA and mRNA systems. Such pioneer and novel imaging platforms can be also generally adapted to study other RNA modifications.